Anti-cancer therapy agent of arsenic hexoxide (As4O6) of a natural chemical substance and its pharmaceutical composition

ABSTRACT

This invention is about the identification of the HD-2, a natural chemical substance that was separated and purified from a natural product, Sinsuk, as arsenic hexoxide (As 4 O 6 ) and about its therapeutic efficacy as an anti-cancer drug and pharmaceutical composition.  
     Arsenic hexoxide (As 4 O 6 ), a natural chemical substance obtained from Sinsuk after eliminating the toxic property, has a potent anti-cancer efficacy by its direct cytotoxicity on tumor cells and suppresses the formation of new blood vessels of tumor masses, which results in complete cure of malignant cancers.

FIELD OF THE INVENTION

[0001] The present invention relates to the identification of HD-2, anatural chemical substance that was separated and purified from anatural product, Sinsti, as arsenic hexoxide (As₄0₆) and about its-therapeutic efficacy as an anti-cancer drug and pharmaceuticalcomposition, and more particularly, to the purification processes of anatural chemical substance (arsenic hexoxide, As₄ O₆) from Sinsuk whileeliminating the toxicity and the novel anti-cancer effect of As₄O₆ andits pharmaceutical composition by their direct cytotoxicity andsuppression of new angiogenesis at and around tumor sites.

BACKGROUND OF THE INVENTION

[0002] In general, various drugs are presently available for anti-cancerchemotherapy. Alkylating agents, such as cisplatin and cyclophosphamide,manifest their anti-cancer effect by forming covalent bonds withnitrogen atoms of DNA nucleotides because of its highly electrophilicproperty of the active site. Antimetabolites, such as 5-fluorouracil,act by inhibiting enzymes involved in biosynthesis of nucleic acids orby being inserted into DNA or RNA structures by itself. Someantibiotics, such as adriamycin, act potently on DNA to inhibit thenormal function, which results in suppression of tumor growth. But allof these anti-cancer agents affect not only pathological tumor cells,but also normal healthy cells, especially bone marrow cells orintestinal epithelia with high turnover rate, which cause seriouscomplications and toxicity, such as myelosuppression, alopecia, renalfailure, nausea and vomiting, neurotoxicity, etc.

[0003] On the other hand, arsenic has been known as a potent,environmental carcinogen, affecting skin and lung often. Arsenic isreported to bind to sulfohydryl structure of enzymes to inactivatetarget enzymes, to inhibit phosphorylation and dephosphorylationreactions, which are vital for regulation of enzyme activities, and tocause abnormalities in chromosomes. Therefore arsenic has been studiedmostly from toxicological aspect, related to these reports up to now.

[0004] But in the past, arsenic had been used as a therapeutic agent inboth Oriental and Occidental medicines. Especially, in traditionalChinese medicine including Korean medicine, arsenic compound had beenprescribed for a long time to treat some fatal diseases, e.g. toeradicate evil energy. In old medical literatures of Korea and China, itis described that arsenic was prescribed as a medicine by the name ofEungwhang in page 1234 or by the name of Bisang in page 1237 ofTonEuiBoGam (NamSaDang), or Encyclopedia of Oriental Medicine, where itis described that arsenic was prescribed only after reducing itstoxicity, because of its extreme toxicity. Also arsenic was known tohave detoxifying activity against several toxic substances. For example,arsenic was used in managing choongak, or vomiting and in eradicatingspirits and evil energy. In an old literature of Chinese medicine(BonChoKangMok (Encyclopedia of Herbs of Chinese Medicine), pages 12-16of vol. 9), indications and pharmacological actions of arsenic (by thename of whangwoong) are described, where arsenic is reported to have theaction of purifying the blood. Thus arsenic had been recognized as anactive medicine and used for a long time, but in Korea, arsenic isrecognized as a possibly harmful chemical with characteristics of heavymetals and accordingly its use is quite limited. Arsenic possesses somecharacteristics of heavy metals although it does not belong to heavymetal group and therefore, has been avoided in the production ofmedicine. Exposure to arsenic leads to anemia, leukopenia, anddysfunction of kidney and liver and chronic exposure may havecarcinogenic effect.

[0005] In Western medicine, arsenic compound was prescribed for treatingseveral diseases, including rheumatism, syphilis, psoriasis, etc. andlow dose of arsenic compound had been known to have beneficial effect onphysiological functions of human body, including stimulation ofhematopoiesis, which coincides with descriptions in old literatures -ofOriental medicine. But in modern medicine, indications for arseniccompound became very limited. From the end of 19th century to thebeginning of 20th century, arsenic compound was tried to treat chronicleukemia and after 1950s, melarsoprol, an organic compound of arsenicwhich was prescribed for African trypanosomiasis, is the only arseniccompound in use at present time.

[0006] Based on these pharmacological properties of arsenic, attemptshave been made recently to develop a novel anticancer drug and presentlysome studies are making rapid progress in this field. After the CulturalRevolution, China has been putting considerable efforts to studytraditional medicine using the scientific tools of Western medicine.They published a report in 1996, in collaboration with a French researchteam, that arsenic trioxide, (As₂O₃) had an excellent effect in treatingacute promyelocytic leukemia. Researchers of Western medicine weremarveled at this result, because arsenic trioxide was especiallyeffective in treating leukemia patients who had been resistant toconventional chemotherapy since this paper was published, more medicalscientists of Western Hemisphere became interested in the possibleanticancer effect of arsenic compounds. Stimulated by these results,considerable efforts have been made to integrate traditional OrientalMedicine and modern molecular medicine by interpreting the results ofOriental Medicine in terms of mainstream modern anticancer chemotherapy.It is extremely important to develop novel chemicals to have effectiveanticancer efficacy without any serious side effects. The inventiondescribed here succeeded in the separation and purification of theactive ingredient by treating a natural, raw material of arsenic, whichhad been used in Oriental medicine, through multiple processes.Additionally clinical study indicated that pharmaceutical composition ofarsenic hexoxide shows potent anticancer efficacy without any obviousside effects.

SUMMARY OF THE INVENTION

[0007] Accordingly, the present invention is to provide a novel, naturalchemical substance, arsenic hexoxide (As₄O₆) obtained from Sinsuk, whileeliminating toxicity.

[0008] The other object of this invention to elucidate the actionmechanism of anticancer efficacy of the novel, natural chemicalsubstance obtained from Sinsuk.

[0009] Another object of the invention is to describe the usage of thenovel, natural chemical substance for anticancer therapy and itspharmacological composition.

[0010] To achieve these advantages in accordance with the purpose of thepresent invention, as embodied and broadly described, first we separatedand purified a natural chemical substance, HD-2, by repeated heating ofSinsuk containing arsenic to eliminate the toxicity, which was followedby structure analysis. The white substance obtained by this procedurewas tested on cloned tumor cells of mice and human beings, to evaluatethe anticancer efficacy of the substance and to see whether theanticancer effects are caused by tumor cell death by apoptosismechanism. Toxicity of HD-2 following acute administration was evaluatedby observing clinical symptoms of rats after a single, oraladministration of a large dose and The toxicity of HD-2 followingsubacute administration was evaluated by observing clinical symptoms ofrats after a slow oral administration. Clonal tumor cells, targeted tolungs, were injected intravenously into mice and HD-2 was administeredorally or through intravenous route. Afterward the number of metastatictumor masses in lungs was counted to evaluate the inhibitory effect ofthe substance on cancer metastasis. Similarly, melanoma cells wereinoculated intradermally into mice, followed by oral administration ofHD-2, after which anticancer mechanism of HD-2 was investigated bycounting the number of new blood vessels formed at or around tumormasses. Cancer was induced by injection of carcinogen into mice andtumor-suppressing efficacy was measured in these mice after oraladministration of HD-2. We also tested a pharmacological compositionprepared by mixing various herbs of Chinese medicine with arsenichexoxide, which was administered orally to cancer patients at terminalstage to evaluate the anticancer efficacy.

[0011] In accordnace with one aspect, the present invention provides ananti-cancer agent of arsenic hexoxide (As₄O₆) of a natural chemicalsubstance and its pharmaceutical composition comprising:

[0012] 1) We separated and purified a natural chemical substance ofwhite color, HD-2, by repeated heating of Sinsuk containing arsenic andarsenic of reagent grade, which were followed by structure analysis toshow that it corresponds to arsenic hexoxide, As₄O₆.

[0013] 2) A natural chemical substance, As₄O₆, obtained by thisprocedure was added to culture media to grow cloned tumor cells of miceand human beings, to evaluate the anticancer efficacy of the substance.

[0014] 3) The anticancer mechanism of As₄O₆ was studied to examinewhether the anticancer efficacy was due to tumor cell death by apoptosismechanism.

[0015] 4) Different amounts of a natural chemical substance, As₄O₆, wasacutely administered orally to male and female rats, to examine theacute toxicity of arsenic hexoxide by observing manifestedcomplications.

[0016] 5) Same amount of a natural chemical substance, As₄O₆, was slowlyadministered orally to male and female rats, to examine the subacutetoxicity of the invention by observing manifested complications.

[0017] 6) Clonal tumor cells, targeted to lungs, were injectedintravenously. into mice and a natural chemical substance, As₄O₆, wasadministered orally or through intravenous route., Afterwards the numberof metastatic tumor masses appearing in lungs was counted to evaluatethe inhibitory effect of the substance on cancer metastasis.

[0018] 7) Similarly, malignant melanoma cells were inoculatedintradermally into mice, folowed by oral administration of a naturalanticancer agent, As₄O₆. Afterwards anticancer mechanism was studied bymeasuring the size of tumor masses and by counting the number of newlyformed blood vessels at and around tumor masses.

[0019] 8) Carcinogen was injected into mice to induce malignant tumorsand anticancer effects of a natural anticancer agent, As₄O₆, werestudied by measuring the incidence and size of tumors in liver ad lung.

[0020] 9) We also prepared pharmaceutical composition by adding variousherbs of Oriental medicine to a natural anticancer agent, AS₄O₆, inseveral forms (tablet, capsule, and solution).

[0021] 10) Tablets prepared as described above were administered orallyto cancer patients at terminal stage, carrying a malignant cancer ofuterus, lung, maxillary sinus, kidney, or urinary bladder, to evaluatethe therapeutic efficacy of As₄O₆. The size of the tumors and clinicalcourses were monitored using computed tomography (CT) and magneticresonance imaging (MRI).

[0022] It is to be understand that both the forgoing general descriptionand the following detailed description are exemplary and explanatory andare intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE ATTACHED DRAWINGS

[0023] The accompaned drawings, which are included to provide a furtherunderstanding of the invention and are incorporated in and constitute apart of this specification, illustrate embodiments of the invention andtogether with the description serve to explain the principles of thedrawings:

[0024] In the drawings:

[0025]FIG. 1 shows schematized procedures for separation and chemicalpurification of Sinsuk.

[0026]FIG. 2 shows the 3-dimensional structure model of Sinsukdetermined by structure analysis.

[0027]FIG. 3 shows the time course of anti-cancer efficacy of Sinsuk(arsenic hexoxide, As₄O₆) in vitro.

[0028]FIG. 4 shows the result of agarose-gel electrophoresis indicatingthat the anti-cancer effect of a natural chemical substance, As₄O₆, isdue to apoptosis effect.

[0029]FIG. 5 shows the inhibitory effect of A₄O₆ on neovascularizationin tumor mass.

[0030]FIG. 6 shows that As₄O₆ decreases hepatoma incidence induced by acarcinogen (NDEA).

[0031]FIG. 7 shows that As₄O₆ decreases the incidence of lung cancerinduced by a carcinogen (NDEA).

[0032]FIG. 8 is a CT (Computed Tomography) scan showing multiple tumormasses in uterus.

[0033]FIG. 9 is a similar CT scan as FIG. 8, indicating multiple tumorgrowth in uterus.

[0034]FIG. 10 is a CT scan of an enlarged uterus due to the invasion oftumor cells at the terminal stage of uterine carcinoma.

[0035]FIG. 11 is another CT scan of the same patient taken at adifferent angle.

[0036]FIG. 12 is a CT scan of a uterus at terminal stage of uterinecarcinoma, which shows several air shadows reflecting perforations onthe uterine wall This indicates disappearance of tumor mass followingadministration of As₄O₆.

[0037]FIG. 13 is a CT scan of a patient with uterine carcinoma atterminal stage, which shows similar findings as in FIG. 12.

[0038]FIG. 14 is a CT scan of a patient with uterine carcinoma atterminal stage, which manifests similar findings as in FIG. 13.

[0039]FIG. 15 is a CT scan of a patient with uterine carcinoma atterminal stage, which manifests similar findings as in FIG. 14.

[0040]FIG. 16 is an MRI (Magnetic Resonance Imaging) scan of uterusfilled with fecal material leaked from rectum through the opening ofuterine perforation, which was formed after the disappearance of cancermass.

[0041]FIG. 17 is an MRI image manifesting similar findings as FIG. 16.

[0042]FIG. 18 is an MRI image of a uterus after the cure of tumor mass.

[0043]FIG. 19 is an MRI image of a patient with uterine carcinoma atterminal stage, which manifests similar findings as in FIG. 18.

[0044]FIG. 20 is an MRI image of a patient with lung cancer at terminalstage, showing pleural fluids filling the right pleural cavity caused byright lung cancer.

[0045]FIG. 21 is a CT scan of a patient with lung cancer at terminalstage, showing irregular tumor mass at right lung.

[0046]FIG. 22 is a CT scan of a patient with lung cancer at terminalstage, showing enlarged lymph nodes in mediastinum.

[0047]FIG. 23 is a CT scan of the same patient as in FIG. 22.

[0048]FIG. 24 is a CT scan of the same patient as in FIG. 23.

[0049]FIG. 25 is a CT scan of a patient with lung cancer at terminalstage, indicating that pleural fluid in right pleural cavity started toshrink in volume following the administration of pharmaceuticalcomposition of As₄O₆.

[0050]FIG. 26 is a CT scan of a patient with lung cancer at terminalstage, showing that pleural fluid in right pleural cavity was completelyabsorbed following the administration of pharmaceutical composition ofAs₄O₆.

[0051]FIG. 27 is a CT scan of a patient with lung cancer at terminalstage, showing the shrinkage of lymph node to normal size following theadministration of pharmaceutical composition of As₄O₆.

[0052]FIG. 28 is a CT scan manifesting the same findings as in FIG. 27.

[0053]FIG. 29 is a CT scan manifesting the same findings as in FIG. 28.

[0054]FIG. 30 is a CT scan manifesting the same findings as in FIG. 29.

[0055]FIG. 31 is a CT scan of a cancer patient involving maxillary sinusat terminal stage, showing that right maxillary sinus was filled withtumor masses.

[0056]FIG. 32 is a CT scan of the same patient as in FIG. 31, taken at adifferent angle.

[0057]FIG. 33 is a CT scan of a patient with a cancer involvingmaxillary sinus, who was being treated for the cancer at a hospital.

[0058]FIG. 34 is a CT scan of the same patient as in FIG. 33.

[0059]FIG. 35 is a CT scan of a patient with a cancer involvingmaxillary sinus at terminal stage, showing that cancerous masses inright nasal cavity and maxillary sinus were cured following theadministration of pharmaceutical composition of As₄O₆.

[0060]FIG. 36 is a CT scan manifesting the same findings as in FIG. 35.

[0061]FIG. 37 is a CT scan manifesting the same findings as in FIG. 36.

[0062]FIG. 38 is a CT scan manifesting the same findings as in FIG. 37.

[0063]FIG. 39 is an IVP (IntraVenous Pyelogram) of a patient with kidneycancer at terminal stage, showing a tumor mass located at left renalpelvis.

[0064]FIG. 40 is an IVP of the same patient as in FIG. 39.

[0065]FIG. 41 is IVP of a patient with kidney cancer at terminal stage,showing a tumor mass located in left renal pelvis growing toward renalartery.

[0066]FIG. 42 are CT scans, taken at different angles, of both kidneysof a patient with kidney cancer at terminal stage.

[0067]FIG. 43 are CT scans of a patient with renal carcinoma at terminalstage, demonstrating the same landings as in FIG. 42.

[0068]FIG. 44 are CT scans of a patient with renal carcinoma at terminalstage, demonstrating the same findings as in FIG. 43.

[0069]FIG. 45 is a CT scan of a patient with renal carcinoma at terminalstage, showing the shrinkage of a cancerous mass following theadministration of pharmaceutical composition of As₄O₆.

[0070]FIG. 46 is a CT scan of a patient with renal carcinoma at terminalstage, demonstrating the same findings as in FIG. 45.

[0071]FIG. 47 is a CT scan of a patient with renal carcinoma at terminalstage, demonstrating the same findings as in FIG. 46.

[0072]FIG. 48 is a CT scan of a patient with renal carcinoma at terminalstage, demonstrating the same findings as in FIG. 47.

[0073]FIG. 49 is a CT scan of a patient with renal carcinoma at terminalstage, showing a marked shrinkage of a cancerous mass in left kidney,following the administration of pharmaceutical composition of As₄O₆.

[0074]FIG. 50 is a CT scan of a patient with renal carcinoma at terminalstage, showing further shrinkage of tumor mass than in FIG. 49.

[0075]FIG. 51 is a CT scan of a patient with renal carcinoma at terminalstage, showing that white-shadowed contrast material filled the spacepreviously occupied by tumor mass in left kidney.

[0076]FIG. 52 is a CT scan of a patient with renal carcinoma at terminalstage, showing tiny tumor masses remaining in left kidney and left renalpelvis.

[0077]FIG. 53 is a CT scan of a patient with urinary bladder cancer atterminal stage, demonstrating tumor masses in dark shadow, located atthe right corner and on the left wall of the urinary bladder.

[0078]FIG. 54 is a CT scan manifesting the same findings as in FIG. 53.

[0079]FIG. 55 is a CT scan of a patient with bladder cancer at terminalstage, manifesting the same findings as in FIG. 54, showing a tumor massin white shadow on left bladder wall.

[0080]FIG. 56 is a CT scan manifesting the same findings as in FIG. 55.

[0081]FIG. 57 is a CT scan of a patient with bladder cancer at terminalstage, showing the disappearance of tumor masses following theadministration of pharmaceutical composition of As₄O₆.

[0082]FIG. 58 is a CT scan of a patient with bladder cancer at terminalstage, manifesting the same findings as in FIG. 57.

[0083]FIG. 59 is a CT scan of a patient with bladder cancer at terminalstage, showing that after treatment, urinary bladder appeared normal.

[0084]FIG. 60 is a CT scan manifesting the same findings as in FIG. 59.

[0085]FIG. 61 is a CT scan manifesting the same findings as in FIG. 60.

[0086]FIG. 62 is a CT scan manifesting the same findings as in FIG. 61.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0087] Reference will now be made in detail to the preferred embodimentsof the present invention, examples of which are illustrated in theaccompanied drawings.

EXAMPLE 1 Separation and Purification of A Natural Chemical Substance,HD-2

[0088] Mixture of 10-g Sinsuk and 10-ml ethanol (C₂H₅OH) was heated for1 hour and then, cooled to room temperature for 1 hour. Another volumeof 10-ml ethanol was added to cooled Sinsuk and the sequential heatingand cooling were repeated several times. The product of this procedurewas washed in 20-ml distilled water with 10-min stirring and shaking for10 min, and 2-ml of distilled water was added to it. 1 min later,precipitates were collected by decanting. This collection process wasrepeated three times. After storing the washed precipitates at −40° C.for 24 hours, precipitates were defrosted and poured onto a filter paperand were dried at room temperature 9 grams of white substance wasobtained as a purified final product.

[0089] The white substance was further purified for detoxification. Saltwas placed in a china made of Kaolin and heated to remove watercomponent. After cooling at room temperature, the white substance wasplaced on the top of the salt and sealed with a filter paper and heatedover 1 hour. After cooling at room temperature, the while sutbstance wascollected, This process was repeated more than 2 times. Finally 2 gramsof a white substance was obtained, which was named as-HD-2 (see FIG. 1).

EXAMPLE 2 Structural Analysis of A Natural Chemical Substance, HD-2

[0090] White substance obtained in EXAMPLE 1 was sent to KoreanInstitute of

[0091] White substance obtained in EXAMPLE 1 was sent to KoreanInstitute of Science and Technology for the structural analysis, whereit was identified as a substance with an empirical formula of As₄O₆ with3-dimensional structure shown in FIG. 2. Physical and chemicalparameters of As₄O₆ are summarized in Table 1. Atomic coordinates (×10⁴)and equivalent isotropic displacement parameters (A²×10³) are listed inTable 2, bond lengths (A) and bond angles (degree) in Table 3, andanistropic displacement parameters in Table 4. TABLE 1 Crystal data andstructure refinement of As₄O₆ parameters Empirical formula As₄O₆ Formulaweight 395.68 temperature 293(2)K wave length 0.71073A Crystal systemcubic Space group Fd3barm Unit cell dimensions a = 11.0457(11)A alpha =90 deg b = 11.046(2)A beta = 90 deg c = 11.0457(10)A gamma = 90 degVolume 1347.7(3) A³ Z 8 Density(calculated) 3.900 Mg/m³ Absorptioncoefficient 19.634 mm⁻¹ F(000) 1440 Theta range for data collection30.98 deg at 3.19 Index range 0 < = h < = 10, 0 < = k < = 14, 0 < = 1 <= 16 Reflections collected 319 Independent reflections 95[R(int) =0.0791] Refinement method Full-matrix least-squares on F²Data/restraints/parameters 95/0/9 Goodness-of-fit on F² 1.009 Final Rindices[I > 2sigma(I)] R1 = 0.0383, wR2 = 0.1111 R indices (all data) R1= 0.0401, wR2 = 0.1130 Absolute structure parameter 10(10) Extinctioncoefficient 0.0039(8) Largest diff. peak and hole 1.056 and −0.865 e.A⁻³

[0092] TABLE 2 Atomic coordinates(×10⁴) and equivalent isotropicdisplacement parameters (A² × 10³) for As₄O₆ As-O#1 1.781(3) As-O#21.781(3) As-O 1.781(3) O-As#3 1.781(3) O#1-As-O#2 98.1(3) O#1-As-O98.1(3) O#2-As-O 98.1(3) As-O-As#3 129(4)

[0093] TABLE 3 Bond lengths [A] and angles [deg] for As₄O₆ X Y Z U(eq)As 1471(1) 6471(1) 8529(1) 13(1) 0 2500 5778(6) 7500 13(2)

[0094] TABLE 4 Anisotropic displacement parameters (A² × 10³) for As₄O₆U11 U22 U33 U23 U13 U12 As 13(1) 13(1) 13(1) 2(1) 2(1) −2(1) O 13(3)14(4) 13(3) 0 5(3) 0

EXAMPLE 3 Anticancer Effect of HD-2 on Cloned Tumor Cells in Vitro

[0095] A natural chemical substance HD-2, obtained in EXAMPLE 1, wasevaluated for anticancer efficacy by exaning -direct cytotoxicity, on:cloned -tumor cells in vitro. Cisplatin was used as a control drug.

[0096] Experiment 1: Anticancer Effect of HD-2 on Cloned Tumor Cells ofMice and Human Beings

[0097] Cloned tumor cells of P388 leukemia, L1210 leukemia, L5178Ylymphoma, Colon26-M3.1 carcinoma, and B16-BL6 melanoma, from mice, andK562 leukemia, liver carcinoma HEP-G2, Hs578T breast cancer, AN-3-CAadenocarcinoma, DLD colon carcinoma, and HeLa epitheloid carcinoma, fromhuman beings, were cultured in EMM, DMEM, or RMPI-1640 culture mediacontaining 7.5% fetal bovine serum (FBS), as described in ATCC manual.After plating cloned tumor cells into test wells at a density of1×10⁴/100 μl, various concentrations of HD-2 and cisplatin were added toexamine cytotoxicity of two substances. Tumor cells in test wells wereincubated in 5% CO₂ incubator at 37° C. for 2 days. The anticancerefficacy of two substances are indicated as a concentration of the testsubstance to inhibit the growth of tumor cells by 50% (ED₅₀, 50%Effective Dose), compared with the growth of control tumor cells, whereneither HD-2 nor cisplatin was added. The results (summarized in Table5) indicate that direct cytotoxicity of HD-2 measured at 48 hours ofincubation was 50±+(mean±SD) times as high as cisplatin. TABLE 5Cytotoxic effect (ED₅₀) on cloned tumor cells clone HD-2 cisplatinmurine leukemia P388 0.17 3.58 leukemia L1210 0.16 2.89 lymohoma L5178Y0.06 — melanoma B16-BL6 0.12 4.7 colon Colon26 0.90 5.6 fibroblast 3T30.03 8.0 human leukemia K562 0.11 — liver carcinoma HEP-G2 0.07 5.6breast cancer Hs578T 0.35 — adenocarcinoma AN3CA 0.06 — colon cancerDLD-1 0.21 6.8 carcinoma Hela 0.05 —

[0098] Experiment 2: Anticancer Effect of HD-2 in 3T3-Fibroblast Cells

[0099] For further study of the cytotoxicity of each substance on clonedtumor cells, 3T3-fibroblast cells were cultured in test wells asdescribed in experiment 1. After plating 3T3-fibroblast cells into testwells at a density of 1×10⁴/100 μl, arious concentrations of HD-2 andcisplatin were added to examine the time-courses (2, 4, and 6 hoursafter the addition) of cytotoxicity, which were measured by XTT method.As shown in FIG. 3, cisplatin did not show any cytotoxic effect up to 24hours after the addition, but HD-2 demonstrated cytotoxic effectstarting from 4 hours after the addition. ED₅₀s of HD-2 were 1.10 μ g/mland 0.21 μg/ml at 4 and 6 hours after the addition, respectively, whichsuggest that HD-2 showed an inhibitory effect on tumor growth from thebeginning of the phase. At the stage of 34 hours of treatment, theeffect was also observed in morphological terms. In cisplatin group,partial necrosis of tumor cells or slowing of tumor growth was observedat this time. In contrast, complete necrosis of tumor cells was observedin H group to cause obvious changes in tumor morphology (such asbreakdown of cell walls), which result in the loss of adhesiveness ofcancer cells. This indicates that the direct killing effect of HD-2 ismanifested within a short period of administration, compared with theeffect of conventional chemotherapeutic agents, such as cisplatin. ED₅₀HD-2 after 34 hours of administration was 60 ng/ml, but ED₅₀ ofcisplatin could not be determined, although partial inhibition of tumorgrowth was observed after 24 hours of administration. At the end of theexperiment (48 hours after administration), ED₅₀s were 30 ng/ml and 8μg/ml for HD-2 and cisplatin, respectively. Thus cytotoxicity of HD-2 isabout 270 times as high as that of cisplatin.

EXAMPLE 4 Mechanism of Cytotoxic Effect of HD-2

[0100] Cytotoxicity of HD-2 was further investigated to examine whetherthis effect was related to tumor cell death by apoptosis mechanism.HL-60 cells were seeded at a density of 2×10⁴ cells/ml and adequateconcentration of HD-2 was dissolved in culture media, after whichcisplatin was added to positive control group and culture media withoutcisplatin was added to negative control group. Cells were centrifugedafter 24-hour incubation and precipitated cells were washed withphysiological buffer solution (PBS) and incubated again in a buffersolution (500 mM Tris-Cl (pH 9.0), 20 mM EDTA, 10 mM NaCl, 1% SDS, and500 mg/ml proteinase K) at 50° C. for 24 hours. Total DNA was collectedusing phenol extraction of cell lysate obtained by this treatment andwas loaded on agarose gel plate for electrophoresis. As shown in FIG. 4,DNA segmentation at ˜180 bp, which is a typical finding of apoptosis,was observed at 2.5 to 25 μg/ml concentrations of HD-2.

EXAMPLE 5 Acute Toxicity of HD-2

[0101] The acute toxicity of oral administration of HD-2 was evaluatedaccording to toxicity assessment criteria described in Article 96-8 ofNotice on Food and Drug Safety (Apr. 16, 1994). Rats (Sprague Dawleystrain) were used for animal experiments. Dosage of a single, oraladministration ranged from 0.4 to 1.25 g/kg body weight in male rats and0.4 to 0.625 kg/kg body weight in female rats. General conditions of theanimals, toxic symptoms, and mortality were measured every hour for theinitial 6 hours following the single administration and once a dayafterwards for 14 days. Body weights were measured before starting thestudy, 7 days after the administration, and at autopsy. Expired ratswere studied to find the cause of death at autopsy. At the end of (hestudy all living rats were killed by an overdose of ether anesthesia andmajor organs were examined for pathological findings with the nakedeyes. With maximal dose in male rats (1.25 g/kg body weight), mortalityreached 100% during the study period. With high dose in male rats (0.85g/kg body weight) mortality was 60% and with medium dose (0.8 g/kg bodyweight), mortality was 10%. In female rats, mortality was 100% withmaximum-dose group (0.625 g/kg body weight), 80% in high-dose group(0.62 g/kg body weight), and 40% in medium-dose group (0.58 g/kg bodyweight). Clinical symptoms within 3 days of oral administration rangedfrom dose-dependent depression and dyspnea. Some rats manifesting theseclinical symptoms expired, but others recovered to normal conditionwithin 2 to 3 days of clinical symptoms. Changes in weight did not showany significant difference between the study and the control groups inall subgroups of different dosages. Autopsy of rats expired during thestudy period revealed findings of expanded stomach and engorged liver.Significant findings related to HD-2 administration were not observed inautopsy of killed rats at the end of the study.

[0102] With oral administration of HD-2 in Sprague-Dawley rats, LD₅₀(50% Lethal Dose) was 0.81 g/kg body weight in male rats, and 0.58 g/kgbody weight in female rats. The results are summarized in Table 6. TABLE6 Mortality of male and female Sprague-Dawley rats following oraladministration of HD-2 Dose Hours after (g/kg treatment Days aftertreatment Final sex B.W.) 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14mortality male 1.25 0 0 0 0 0 0 3 1 0 0 0 0 0 0 0 0 0 0 0 0 5/5(100%)0.85 0 0 0 0 0 0 2 1 0 0 0 0 0 0 0 0 0 0 0 0 3/5(60%) 0.8 0 0 0 0 0 0 11 0 0 0 0 0 0 0 0 0 0 0 0 2/5(10%) 0.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0/5(0%) 0.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5(0%) 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5(0%) Female 0.625 0 0 0 0 0 0 1 00 0 0 0 0 0 0 0 0 0 0 0 5/5(100%) 0.62 0 0 0 0 0 0 3 1 0 0 0 0 0 0 0 0 00 0 0 4/5(80%) 0.58 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 2/5(40%) 0.50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5(0%) 0.4 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0/5(0%) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00/5(0%)

EXAMPLE 6 Subacute Toxicity of HD-2

[0103] The subacute toxicity of oral administration of HD-2 wasevaluated according to toxicity assessment criteria described in Article96-8 of Notice on rood and Drug Safety (Apr. 16, 1994). Rats (SpragueDawley . strain) were used for experiments, as was the case for acutetoxicity experiments. Dosage of oral administrations was 100 (highdose), 10 (medium dose), and 1 mg (low dose) per kg body weight, whichwere administered once a day for 4 weeks (total 28 administrations).

[0104] Following items were observed during the period of study.

[0105] 1) General symptoms: General symptoms, such as anorchism,salivation, diarrhea, vomiting, polyuria, anuria, and fecal change andthe severity of these symptoms were evaluated once a day during thestudy period.

[0106] 2) Food consumption: Twice a week, the amount of consumed foodand the remaining amount were checked per cage.

[0107] 3) Water consumption: Twice a week, the amount of consumed waterand the remaining amount were checked per cage.

[0108] 4) Weight: Weights were measured twice a week until the end ofthe study.

[0109] 5) Urinalysis: Urine samples were collected during study periodfrom 5 randomly selected rats per study subgroup and appearance, volume,and colors were recorded. Using urinalysis kits (N-multistix ofAmersham), pH, gravity, leukocyte, protein, ketone body, urobilinogen,glucose and blood urea nitrogen were measured.

[0110] 6) Eye examination: Ophthalmoscopic examination of 5 randomlyselected rats per study subgroup was performed to evaluate the externalappearance, cornea, and:fundus of the eye.

[0111] 7) Hematological and biochemical analysis: Routine blood test wasdone to measure red blood cell count, white blood cell count, hemoglobinconcentration, number of monocytes and lymphocytes, and bloodcoagulation time. Biochemical analysis of serum was done to measure theactivity of albumin transferase, aspartate transaminase, alkalinephosphate, and albumin.

[0112] 8) Size and weight of organs: For every animal studied, weightand size of major organs were measured relative to body weight. Measuredorgans included liver, kidney, spleen, heart, adrenal gland, brain,thyroid gland, ovary, and testis.

[0113] 9) Pathological examination: Organs were fixed in formalin aftermeasurement of weight and size and fixed tissues were cut into 5-mmslices using microtome (AO Rotate Microtome) and stained withhematoxylin and eosin for microscopic study.

[0114] During the study, fatal cases were not observed and no specificclinical symptom, including changes in weight and consumption of foodand water, was observed. Also any significant abnormality was notobserved on urinalysis mad eye examination. Hematological andbiochemical study did not reveal any significant difference between thestudy and the control groups. On pathological examination at autopsy,hemosiderin located in the cytoplasm of proximal tubular epithelium andatrophy of proximal tubular epithelium of kidney were observed to aslight degree in high-dose group (100 mg/kg body weight), but not inmedium-dose, low-dose, and control groups. Other than this, nopathological finding to have dose-dependent property or to be related toHD-2 administration was observed. These results are summarized in Table7. Therefore, oral administration of HD-2 lasting for 4 weeks did notcause any significant hematological abnormality in high-dose group (100mg/kg body weight), but mild pathological finding suggestive of a slightrenal abnormality was observed. However in medium-dose group, no suchpathology was observed. TABLE 7 Biochemical parameters of female ratstreated with oral dose of HD-2 /Group Control Low Medium HighParameter/Dose(g/kg/day)/ 0 0.03 0.3 3.0 No. of animal 10 10 10 10 ALT37.66^(a) 27.10 27.00 29.67 (μ/l) ±7.91 ±11.63 ±9.42 ±10.55 AST 118.5370.10* 87.53 77.87* (μ/l) ±20.23 ±30.11 ±11.89 ±23.89 ALP 67.00 24.0038.00 34.00 (μ/dl) ±52.37 ±7.00 ±8.76 ±14.73 CREAT 0.37 0.40 0.53 0.47(mg/dl) ±0.15 ±0.17 ±0.15 ±0.15 BUN 13.00 11.43 13.18 13.13 (mg/dl)±2.26 ±4.28 ±3.62 ±4.60 ALB 3.27 3.60 3.70 3.33 (g/dl) ±1.27 ±1.54 ±1.16±0.99 GLU 50.33 67.70 70.00 63.03 (mg/dl) ±30.86 ±46.60 ±32.04 ±40.83 TB0.19 0.14 0.16 0.18 (mg/dl) ±0.07 ±0.05 ±0.07 ±0.01 Ca 8.30 8.77 8.958.73 (mg/dl) ±3.03 ±3.18 ±2.64 ±3.25 CL 90.00 87.33 91.00 88.33 (meq/dl)±26.00 ±28.01 ±23.47 ±26.31 TP 5.87 6.27 6.53 5.93 (g/dl) ±2.05 ±2.24±1.89 ±2.11 CHOL 40.33 42.33 40.50 44.33 (mg/dl) 17.79 ±21.01 ±11.36±13.01 TG 46.00 50.00 56.25 62.33 (mg/dl) ±27.06 ±17.35 ±29.49 ±20.53

EXAMPLE 7 Effect of HD-2 on Cancer Metastasis

[0115] Experiment 1: Inhibitory Effect of Orally-Administered HD-2 onCancer Metastasis

[0116] Utilizing mouse model, the inhibitory effect of HD-2 on cancermetastasis was evaluated with cloned tumor cells and compared withcisplatin. As single administration of 500 mg/kg body weight per day didnot have any side effect in rats (see EXAMPLE 5), inhibitory effect ofHD-2 on cancer metastasis was studied employing dose below 500 mg/kgbody weight. B16-BL6 melanoma cells or colon26-M3.1 carcinoma cells wereinoculated into mice and number of metastatic tumor masses appearing inlungs was counted. After the inoculation of tumor cells, various dosesof of HD-2 or cisplatin were administered one day after the inoculationto find the optimum concentration for anti-metastatic efficacy. Sevendays after the inoculation, HD-2 or cisplatin was administered tomeasure therapeutic efficacy on grown tumor mass. As shown in Table 8,oral administration of HD-2 (0.1 to 10 mg) had significantanti-metastatic effect compared to the control group (cisplatin group).The peak activity was observed a: 1-mg dose with very high anticancerefficacy (86%). A 7th day when inoculated tumor cells settled completelyin target organs, oral administration of HD-2 demonstratedanti-metastatic efficacy of 70%. This indicated oral administration ofHD-2 was quite effective for treatment of established cancer. TABLE 8Inhibitory effect of orally-administered HD-2 on cancer metastasisConcentration, number of metastatic masses administration (inhibitoryrate(%) route & day mean ±SD range Experiment I. 122 ± 20 101-146Control group (injection of B16-BL6) HD-2 10 mg oral administration +145 ± 25(63.1) 72-23 1 mg oral administration +1 17 ± 9*86.1) 8-29 0.1 mgoral administration 75 ± 28(38.5) 105-51 +1 Experiment II. 162 ± 24133-188 Control group (injection of B16-BL6) HD-2 10 mg oraladministration +7 55 ± 13(66.1) 40-67 1 mg oral administration +7 48 ±19(70.4) 26-69 0.1 mg oral administration 95 ± 23(41.4) 118-72 +7

[0117] Similar to Experiment 1, the inhibitory effect of HD-2 on cancermetastasis was compared with cisplatin, using cloned tumor cellspossessing high metastatic capability. In this experiment, HD-2 wasadministered intravenously with dosage less than 500 mg/kg body weightper day. As summarized in Table 9, 10-to 100-μg HD-2 had anti-metastaticefficacy above 90%, which suggested that HD-2 was more effective thancisplatin at the same dose. Ten micrograms of HD-2 and cisplatin, whichis considered as a optimum dose to inhibit cancer metastasis at 7th dayof tumor cell inoculation, had anticancer effect of 67.5% and 50.0%,respectively, when administered intravenously. This suggests that theanticancer efficacy of HD-2 is better than the conventional anticancerdrugs and HD-2 is also effective in treating full-grown cancer atterminal stage. TABLE 9 Inhibitory effect of intravenously-administeredHD-2 on cancer metastasis Concentration, number of metastatic massadministration (inhibitory rate(%) route and day mean ± SD rangeExperiment I. Control group (injection of colon tumor cells) 155 ± 26122-179 HD-2 100 μg intravenous administration +1 15 ± 15(90.3) 2-28 10μg intravenous administration +1 11 ± 19(92.9) 1-29 1 μg intravenousadministration +1 52 ± 28(66.5) 27-71 cisplatin 100 μg intravenousadministration +1 57 ± 22(63.2) 32-80 10 μg intravenous administration+1 20 ± 13(87.1) 8-32 1 μg intravenous administration +1 102 ± 28(34.2)84-127 Experiment II. Control group (injection of colon tumor cells) 154± 14 142-167 HD-2 10 μg intravenous administration +7 50 ± 11(67.5)39-65 cisplatin 10 μg intravenous administration +7 77 ± 12(50.0) 5-88

EXAMPLE 8 Anticancer Mechanism of HD-2 in vivo

[0118] The in vivo mechanism of anticancer effect of HD-2 was studied inmice. After suspending 4×10⁵ cells of B16-BL6 melanoma in 50% PBS, theywere injected intradermally into 2 sites on the back of 6- to 7-week oldC57BL/6 mice. Three days after the tumor injection, one milligram ofHD-2 was given orally and size of inoculated melanoma and number ofblood vessels at and around tumor sites were measured. Control group wastreated with oral administration of PBS. As demonstrated in FIG. 5,number of new blood vessels, which are observed in cancer proliferationand metastasis, tended to decrease following the administration of HD-2.Also the size of solid tumor mass decreased significantly in proportionto decrease in the number of new blood vessels. It is suggested thatHD-2 suppresses the invasion into and the adhesion onto tissues, whichgoes hand in hand with the formation of new blood vessels.

EXAMPLE 9 Inhibitory Effect of HD-2 on Carcinogen-Induced Oncogenesis

[0119] To examine the inhibitory effect of HD-2 on carcinogen-inducedoncogenesis, N-nitrosodiethylamine (NDEA) was injected as a carcinogeninto peritoneal cavity of mouse (B6C3F1 strain) at a concentration of 90mg per kg body weight to induce cancer. At 2, 4, 8, 16, and 32 weeksafter carcinogen injection, 100 g of HD-2 was administered orally andthe same amount of distilled water was injected into control group.Forty-two weeks after NDEA treatment, mice were sacrificed to measurethe incidence and the size of tumors formed in lung and liver. As shownin FIG. 6, the incidence of NDEA-induced hepatic tumor was effectivelyinhibited after oral administration of HD-2. The incidence ofNDEA-induced hepatic tumor was above 90%, but following HD-2administration, the incidence was lowered to 5 to 22%, despite ofvariation depending on the period of HD-2 administration. Thus HD-2inhibited carcinogen-induced oncogenesis in liver by 78 to 95. Also HD-2inhibited spontaneous hepatoma completely, the incidence of which isreported about 20% without HD-2 administration. In lung, the inhibitoryeffect of HD-2 on reducing carcinogen-induced oncogenesis was not sodramatic as in liver. However, if HD-2 was given at 4 weeks after NDEAinjection, carcinogen-induced oncogenesis was inhibited by 30%. Further,spontaneous cancers of lung were completely suppressed by HD-2, whichindicates that oral administration of adequate dose of HD-2 decreasesthe incidence of spontaneous cancers. As shown in FIG. 7, the number oftumor masses in lung was about 2 in HD-2 group, compared to 7 in controlgroup, which points to the efficacy of HD-2 in inhibition ofcarcinogen-induced oncogenesis. These results suggest that HD-2 was veryeffective not only in treatment but also in prevention of malignantcancers.

EXAMPLE 10 Preparation of Pharmaceutical Composition for AnticancerTherapy

[0120] 5 g of HD-2 was mixed with the following ingredients of Chinesemedicine and pulverized to a powder form: hodongjoo 7 g, chunsangap 7 g,baekchool 10 g, woowhang 3 g, sahyang 3 g, shingok 5 g, moryo 5 g,yongnyehyang 3 g, yoohyang, 5 g, molryak 5 g, baekbongryung 10 g,sangbaekpi 10 g, galgeun 10 g, macheehyun 5 g, ohmeeja 5 g, hyulgal 5 g,seokko 5 g, boongsa 5 g, hansooseok 5 g, and red steamed ginseng 7 g.Distilled water was added to the powder to form pills of 1 to 1.5 gramsfor oral administration. These pills were used to manufacture tablets of˜1.33 g convenient for a single dose, which were administered to cancerpatients at terminal stage, three times a day to make a total of 4 gramsper day. The effective dose of HD-2 may depend on the fraction of drugsand age, sex, and health conditions of the patient. In general, usualdosage was 50 g per kg body weight, with upper limit of 160 to 330 g perkg body weight. Although ingredients of Oriental medicine were utilizedto prepare pharmaceutical composition for the clinical trial of HD-2,any pharmaceutical composition can be employed for this purpose.Chemically-synthesized arsenic hexoxide (As₄O₆) can be substituted forHD-2, which was prepared by separation and purification of Sinsuk inthis study.

EXAMPLE 11 Clinical Trial on Various Forms of Malignant Cancers

[0121] Cancer patients diagnosed of cancer of uterus, lung, maxillarysinus, kidney, or urinary bladder at hospital by thorough clinicalexaminations were selected for the study and most were at the terminalstage of the disease with expected survival of 6 to 12 months. Afteracquiring consent from the patient or the guardian, tablets described inEXAMPLE 10 were administered 3 times a day to examine the anticancerefficacy.

[0122] Experiment 1: Clinical Trial on a Patient with Uterine Cancer

[0123] The study subject (EunSook Park) was diagnosed of cancer ofuterine cervix (final diagnosis: squamous cell carcinoma) at SeoulNational University Hospital on October 1993. Even after repeatedanticancer therapy (8 times), cancer cells continued to grow and involvelymph nodes, rectum, and urinary bladder. Therefore urine was collectedthrough a tube inserted into the right kidney and the patient wasimmobilized in bed and incapable of taking food. The doctor informed herof expected survival of less than 3 months. Tablets described in EXAMPLE10 were administered to EunSook Park for 3 months and the progress wasmonitored using computed tomography (CT) and magnetic resonance imaging(MRI). CT scans (FIG. 8 to 19) indicated that following thedisappearance of tumor mass, perforations were formed in the walls ofuterus urinary bladder, ard rectum and feces of rectum leaked intouterus through perforated openings, for which colostomy were done on thepatient on January 1996.

[0124] Experiment 2: Clinical trial on a Patient with Lung Cancer

[0125] The study subject (KyungJoo Lee) was a male of age 30 and treatedfor fever and chill with the diagnosis of pneumonia on Mar. 19, 1996without any improvement of symptoms. He was diagnosed of stage-4 lungcancer (final diagnosis: undifferentiated adenocarcinoma) at SeongGaHospital of Bucheon and confirmed of the diagnosis at Samsung MedicalCenter located at IlWanDong, Seoul with additional thoroughexaminations. The doctors told him of his limited lifetime of 6 to 12months. CT scans (FIGS. 21 to 24), taken on Mar. 21, 1996 at SeongGaHospital, showed irregular tumor mass at the right lung, pleural fluidsfilling the right pleural cavity, and enlarged lymph nodes inmediastinum. Kyungjoo Lee was given the tablets prepared as described inEXAMPLE 10 for 8 months, while the progress of the disease was monitoredusing CT scanning. As indicated in FIGS. 25 to 30, tumor mass graduallyshrank in size to disappear completely after 8 months of drug therapy.

[0126] Experiment 3: Clinical Trial on a Patient with Maxillary SinusCancer

[0127] The study subject (HeeGon Kim) was diagnosed of malignant cancerinvolving right nasal cavity and maxillary sinus (final diagnosis:adenoid cystadenoma) in 1981, which was inoperable due to metastasis tobone. He had been treated with chemotherapy and radiation therapy atCheonju Jesuit Hospital and seoul National Unuversty hospital, but thedisease became worse. He was advised to prepare for his death after a CTscan on Mar. 5, 1990. As shown in CT scans taken on Mar. 31, 1990 (FIGS.31 and 32 ), right maxillary sinus was filled with tumor masses andtumor mass was also observed in right nasal cavity. Cancer specialistsat Seoul National University Hospital prescribed anticancer chemotherapyfor 2 months, but CT scans taken after completing the chemotherapyindicated additional growth of tumor masses to involve nearby brainregions, right eyeball, and right and left nasal cavity. HeeGon Kim wasgiven the tablets prepared as described in EXAMPLE 10 for 3 months andthe progress of the disease was checked using CT scanning on Feb. 27,1991 at Seoul National University Hospital. CT scans (FIGS. 35 to 38)indicated that most of the tumor masses were gone and right nasal cavityand maxillary sinus were filled with normal flow of air.

[0128] Experiment 4: Clinical Trial on a Patient with Kidney Cancer

[0129] The study subject (YongHa Lee) was diagnosed of terminal-stagerenal cancer at urology department of Pusan Merinol Hospital afterthorough examinations including CT scanning. He gave up surgicaltreatment after being told of low survival rate of 20% even with radicalnephrectomy. CT scans taken at dismissal (FIGS. 39 to 44) showed thatthe left kidney appeared enlarged compared to the right one and leftrenal pelvis was not filled with contrast material, indicating tumormass in that region. Intravenous pyelograms were taken afteradministering tablets prepared as described in EXAMPLE 10. Intravenouspyelograms (FIGS. 45 and 46) indicated market decrease of tumor massfollowing 6 months of drug therapy and CT scans (FIGS. 47 to 50)demonstrated 80% decrease of tumor mass. Left nephrectomy was done atPusan Baek Hospital and was confirmed of renal cell carcinoma bypathological examination. With additional administration of tablets asdescribed in

10 for 3 months, CT scans (FIGS. 51 and 52) demonstrated only tiny tumormass located in left kidney and renal pelvis, indicating the disease wasalmost cured.

[0130] Experiment 5: Clinical Trial on a Patient with Urinary BladderCancer

[0131] The study subject (DaeJoong Kim) had been feeling dysuria fromJune 1995 and was treated for cystitis without any improvement. He wasdiagnosed of urinary bladder cancer at Samsung Medical Center withthorough examination including CT scanning. With further study at SeoulJoongAng Hospital, CT scans (FIGS. 53 to 56 ) showed tumor masses indark shadow on the right corner and left wall of urinary bladder and thesurvival rate was estimated about 20% within one year. He was treatedwith tablets prepared as described in EXAMPLE 10 for over 1 year. CTscans (FIGS. 57 and 58), taken at DongIn Hospital of KangNeung on July1996, indicated no evidence of cancer mass and CT scans (FIGS. 59 to62), taken at HyunDaeHospital on Mar. 18, 1997, indicated complete cureof the disease without any shadow of tumor mass.

[0132] As shown in EXAMPLES and experiments described above, arsenichexoxide (As₄O₆), which was obtained by separation and purification froma natural material, Sinsuk, had a potent anticancer efficacy in both invivo and in vitro experiments and inhibited cancer metastasiseffectively in animal experiments. Further the natural arsenic compound(As₄O₆) was mixed with other ingredients of Oriental medicine to maketablets for oral administration. Clinical trial on cancer patientscarrying cancer of uterus, lung, maxillary sinus, kidney, or urinarybladder indicated marked inhibition of proliferation and metastasis ofcancer cells following the administration of tablets made from As₄O₆.This suggests that the invention could be used as an effectiveanticancer drug, which may have great impact on the progress ofbiomedicine.

[0133] It will be apparent to those skilled in the art that variousmodifications and variations can made in an anti-cancer therapy agent ofarsenic hexoxide (As₄O₆) of a natural chemical substance and itspharmaceutical composition of the present invention without departingfrom the spirit or scope of the invention thus, it is intended that thepresent invention cover the modifications and variations of thisinvention provided they come within the scope of the appended claims andtheir equivalents.

1. Use of As₄O₆ as anticancer drug.
 2. Pharmaceutical composition foranticancer therapy consisting of As₄O₆ and other ingredients 3.Pharmaceutical composition according to claims 1 or 2, in which As₄O₆ isseparated and purified from Sinsuk.
 4. Pharmaceutical compositionaccording to claim 2, in which ingredients of Oriental medicine wasused.